Production of fermented needle extracts from red pine and their functional characterization

Pine needle (Pinus densiflora Sieb. et Zucc.) extract is being used for millennia in treatment of multiple ailments in Asia Pacific region. The extract is a composite mixture of a number of organic compounds. Terpenoid is one of such complex compounds that exhibit useful effects. To remove harmful substances with improving functionality of the extract, people adopt traditional spontaneous fermentation followed by aging. To assess the fermentation induced effectiveness we analyzed composition of the volatile terpenoid in fresh to the 7 years old self-fermented extracts along with their anti-bacterial and anti-oxidant properties. Simultaneously, we also analyzed the effect of remarkably changed 1,8-cineole and camphor. SFPEs (self-fermented pine needle extracts), 1,8-cineole, and camphor inhibited growth of bacterial strains like Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Comparative studies between pine needle extracts, 1,8-cineole and camphor showed that the self-fermented pine needle extracts retard the bacterial growth effectively and have relatively higher antioxidant activities. PE (pine needle extract) and SFPEs showed nearly 95% DPPH radical scavenging activities, 1,8-cineole 84% while camphor showed no scavenging activities. The results showed that self-fermentation and aging improves the effects of the extract therefore it could be a good source for nutraceutical product.     

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.     

Antioxidant and antiapoptotic effects of pine needle powder ingestion and endurance training in high cholesterol-fed rats

[Purpose]

Pine needle is a kind of medicinal plant ingested traditionally for a variety of purposes. Therefore, we examined the antioxidant and antiapoptotic capacities of pine needle ingestion in high cholesterol-fed and endurance exercise-trained rats.

[Methods]

Animals were divided into six groups as; CON: normal diet control group; EX: normal diet and exercise training group; HC: high cholesterol diet group; HCE: high cholesterol diet and exercise training group; HCP: high cholesterol and pine needle group; HCPE: high-cholesterol and pine needle diet with exercise training group, respectively. Each group consisted of seven Sprague-Dawley male rats. The swim-training groups, EX, HCE, and HCPE swam in the swim pool 60 min/d and 5 d/week for 5 weeks. During the rearing periods, freeze-dried pine needle powder mix with 5% of the high cholesterol diet was supplied to the HCP and HCPE groups. Gastrocnemius muscle was used as the skeletal muscle. Malondialdehyde (MDA), Mn-containing superoxide dismutase (Mn-SOD), Cu, Zn containing superoxide dismutase (Cu,Zn-SOD), and glutathione peroxidase (GPx) were analyzed for their antioxidant capacities. Finally, p53, Bcl-2 (B-cell lymphoma 2), caspase-3 protein expression was analyzed to determine antiapoptotic ability.

[Results]

MDA showed low content in HCPE compared to the HC. Mn-SOD, Cu,Zn-SOD, and GPx protein expression was significantly increased by pine needle ingestion and/or exercise training. In addition, suppression of p53 protein expression resulted in Bcl-2 increase followed by caspase-3 decrease with/without pine needle ingestion and exercise training.

[Conclusion]

When exercise training in addition to pine needle powder ingestion may be a helpful nutritional regimen to athletes and exercisers.     

Anatomical changes with needle length are correlated with leaf structural and physiological traits across five Pinus species

The genus Pinus has wide geographical range and includes species that are the most economically valued among forest trees worldwide. Pine needle length varies greatly among species, but the effects of needle length on anatomy, function, and coordination and trade‐offs among traits are poorly understood. We examined variation in leaf morphological, anatomical, mechanical, chemical, and physiological characteristics among five southern pine species: Pinus echinata, Pinus elliottii, Pinus palustris, Pinus taeda, and Pinus virginiana. We found that increasing needle length contributed to a trade‐off between the relative fractions of support versus photosynthetic tissue (mesophyll) across species. From the shortest (7 cm) to the longest (36 cm) needles, mechanical tissue fraction increased by 50%, whereas needle dry density decreased by 21%, revealing multiple adjustments to a greater need for mechanical support in longer needles. We also found a fourfold increase in leaf hydraulic conductance over the range of needle length across species, associated with weaker upward trends in stomatal conductance and photosynthetic capacity. Our results suggest that the leaf size strongly influences their anatomical traits, which, in turn, are reflected in leaf mechanical support and physiological capacity.     

培养真菌的试剂松膏和松膏培养基

松树针叶浸提物具有抑制细菌、放线菌等微生物的生长以及促进真菌生长的作用,作者在原工作基础上进一步研制出培养真菌的试剂松膏。按一定比例将松膏加入到培养基中,就可以抑制细菌等非目的微生物,并促进真菌的生长。因此,松膏是为配制选择性真菌培养基和真菌的一般培养用培养基而提供的试剂。     

Pisolithus arhizus ectomycorrhiza affects plant competition for phosphorus between Pinus elliottii and Panicum chamaelonche

 Our objective was to evaluate the ability of an ectomycorrhizal fungus to alter the competitive interaction of pine seedlings growing with grass, and to determine whether the interaction was modified by soil-phosphorus (P) concentration. Slash pine (Pinus elliottii), inoculated with the ectomycorrhizal fungus Pisolithus arhizus or fortuitously colonized by Thelephora terrestris, and a native grass (Panicum chamaelonche) were grown in a greenhouse at three P levels (0.32, 3.22, 32.26 μM H₃PO₄). Pine inoculated with P. arhizus took up more P when competing with the nonmycorrhizal grass than when competing with another pine (irrespective of pine mycorrhizal status). Phosphorus uptake kinetics (C_min, the minimum concentration at which P can be absorbed from a solution; I_max, the maximum uptake rate) for pine and grass were also determined under hydroponic conditions. Pine had a higher I_max than grass but grass had a lower C_min, suggesting that pine is more competitive at higher nutrient concentrations while grass is more competitive at lower nutrient concentrations. The controlled conditions used in these experiments allowed us to evaluate specific parameters (P uptake and absorbing surface area) affecting plant competition. Accepted: 7 August 1999     

Interactions between needles of Pinus resinosa and ectomycorrhizal fungi

Relatively little is known about the factors controlling ectomycorrhizal fungal communities. One possible factor is forest litter chemistry. In a series of experiments we demonstrated that the growth of ectomycorrhizal fungi able to colonize red pine ( Pinus resinosa Ait.) are differentially affected by red pine needles and needle chemical components. For example, water extracts of pine needles stimulated the growth of Suillus intermedius (Smith & Thiers) Smith & Thiers and inhibited the growth of Amanita rubescens Pers. Catechin and epicatechin gallate, components of the water extract, acted similarly to the extract. The volatile compounds α- and β-pinene also had differential effects on the growth of the various species of ectomycorrhizal fungi. Our results suggest that forest litter chemistry has the potential differentially to affect the growth of ectomycorrhizal fungal species and so could affect the structure of ectomycorrhizal fungal communities.     

Anti-oxidant activity and effect of Pinus morrisonicola Hay. on the survival of leukemia cell line U937

The free radical scavenging and anti-cancer activites of Pinus morrisonicola Hay. were studied using different parts of the pine, namely, needle, bark and cone. Results showed that pine needle water extract has the highest scavenging superoxide anion activity and the lowest IC50 value in inhibiting superoxide anion formation; however, the bark water extract showed the best anti-lipid peroxidation activity. Additionally, needle water extract displayed the highest inhibition of leukemia cell line U937 growth. The results indicated that P. morrisonicola Hay. possesses potential chemopreventative and therapeutic properties.     

Novel Lignan and Stilbenoid Mixture Shows Anticarcinogenic Efficacy in Preclinical PC-3M-luc2 Prostate Cancer Model

Prostate cancer is the most common cancer of men in the Western world, and novel approaches for prostate cancer risk reduction are needed. Plant-derived phenolic compounds attenuate prostate cancer growth in preclinical models by several mechanisms, which is in line with epidemiological findings suggesting that consumption of plant-based diets is associated with low risk of prostate cancer. The objective of this study was to assess the effects of a novel lignan-stilbenoid mixture in PC-3M-luc2 human prostate cancer cells in vitro and in orthotopic xenografts. Lignan and stilbenoid –rich extract was obtained from Scots pine (Pinus sylvestris) knots. Pine knot extract as well as stilbenoids (methyl pinosylvin and pinosylvin), and lignans (matairesinol and nortrachelogenin) present in pine knot extract showed antiproliferative and proapoptotic efficacy at ≥40 μM concentration in vitro. Furthermore, pine knot extract derived stilbenoids enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis already at ≥10 μM concentrations. In orthotopic PC-3M-luc2 xenograft bearing immunocompromized mice, three-week peroral exposure to pine knot extract (52 mg of lignans and stilbenoids per kg of body weight) was well tolerated and showed anti-tumorigenic efficacy, demonstrated by multivariate analysis combining essential markers of tumor growth (i.e. tumor volume, vascularization, and cell proliferation). Methyl pinosylvin, pinosylvin, matairesinol, nortrachelogenin, as well as resveratrol, a metabolite of pinosylvin, were detected in serum at total concentration of 7−73 μM, confirming the bioavailability of pine knot extract derived lignans and stilbenoids. In summary, our data indicates that pine knot extract is a novel and cost-effective source of resveratrol, methyl pinosylvin and other bioactive lignans and stilbenoids. Pine knot extract shows anticarcinogenic efficacy in preclinical prostate cancer model, and our in vitro data suggests that compounds derived from the extract may have potential as novel chemosensitizers to TRAIL. These findings promote further research on health-related applications of wood biochemicals.     

In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria

Aims: To investigate the action of different polyphenolic compounds, extracted from red wine, grape marc and pine bark, on oral bacteria. Methods and Results: The anti-microbial activity of extracts was examined by determining the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration using the macro dilution broth technique. Their effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. The effect on glucosyltransferase activity was analysed through the reductions in the overall reaction rate and the quantity of insoluble glucan (ISG) synthesized. Pine bark and grape marc extracts were the most effective inhibitors of the multi-species biofilm formation and of the ISG synthesis. Conclusion: The tested components showed an interesting anti-plaque activity in vitro. Significance and Impact of the Study: This is, to our knowledge, the first and the most complete report on the properties of wine and pine bark extracts that could be used for oral disease prevention purpose.     

Pine Litter from Afforestations Hinders the Establishment of Endemic Plants in Semiarid Scrubby Habitats of Natura 2000 Network

A sowing experiment was carried out using three endemic secondary successional species that commonly co‐occur with Pinus halepensis in afforestations in southeast Spain. The experiment simulated the natural arrangement of needle litter over soil to test the litter's effect on seedling emergence and early growth of dwarf‐shrubs and annual species, the main components of some European Habitats of Community Interest in Spain. Three sowing treatments were compared in a growth chamber: (1) soil without litter taken from clearings in a pine plantation (BARE); (2) intact soil and litter forming two distinct layers as in the field (PINE); and (3) soil and pine litter mixed in the laboratory (PINEMX). Seedling emergence of Diplotaxis harra and Thymus zygis were seven and two times higher, respectively, on BARE than on the pine litter treatments. Pine litter significantly decreased seedling biomass, length, and number of leaves of D. harra and T. zygis. Thymus capitatum was weakly affected by the presence of pine litter. We suggest that plant recruitment in the understory of P. halepensis is partially controlled by interference of pine litter, which might affect dynamics of the studied species. We propose that management practices counteract this negative effect by manipulating the pine litter layer to achieve successful ecological restoration and conservation of endemic‐rich secondary communities in Natura 2000 sites in semiarid western Mediterranean areas.     

马尾松诱导抗性对马尾松毛虫幼虫生长发育的影响

利用不同受害方式和受害程度的马尾松 (PinusmassonianaLamb.)针饲养马尾松毛DendrolimuspunctarusWalker虫幼虫 ,研究受害松针对幼虫生长发育的影响。结果表明 :马尾松毛虫取食受害松针后 ,幼虫的取食量下降 ,排粪量减少 ,对食物的消耗和利用指数降低 ,幼虫发育速率缓慢 ,死亡率增加 ,从而影响马尾松毛虫的种群动态     

Identification and comparative analysis of microRNAs in <Emphasis Type="Italic">Pinus massoniana</Emphasis> infected by <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

Pine wilt disease seriously endangers pine tree growth and can lead to wilting and death. In this study, Masson pine (Pinus massoniana) was used as the donor plant to study miRNA expression by high-throughput sequencing in needle leaves of trees during the first 3 days after infection by Bursaphelenchus xylophilus. The results showed that the number of differentially expressed miRNAs first increased and then decreased in samples within 3 days of infection, with the highest expression in samples collected on day 2. Analysis of miRNA target genes in needles on different days after infection revealed enrichment in two pathways: plant hormone signal transduction and RNA transport. Further comparison of the samples indicated differential expression of 10 miRNAs on different days after infection, and the corresponding target genes, such as those participating in plant hormone signal transduction, were also significantly enriched. In addition, the concentrations of zeatin and indole acetic acid in needle tissues were significantly lower after infection than in the control sample. The above results indicated that B. xylophilus infection influenced the expression of miRNAs related to plant hormone signal transduction in P. massoniana, decreasing plant hormone synthesis and ultimately affecting the growth of Masson pine.     

Pine volatiles mediate host selection for oviposition by Thaumetopoea pityocampa (Lep., Notodontidae)

The oviposition behaviour and host selection by females of the pine processionary moth, Thaumetopoea pityocampa, was studied under two conditions: in a field insectary and in a pine stand. An oviposition choice test was conducted in an insectary cage, using artificial Christmas trees (ACTs) baited with extracts from four pine species: Pinus pinea, Pinus pinaster, Pinus halepensis and Pinus brutia, plus a control. Females oviposited significantly more egg batches on the ACT baited with a P. brutia extract, while no oviposition occurred on the control ACT. In a large P. pinea stand, two groups of randomly selected trees were marked and baited respectively with an extract of P. brutia, which was the preferred species under insectary conditions and with a solvent, to act as control. Results showed that oviposition in the field followed an aggregated pattern, fitting a negative binomial distribution and that trees baited with P. brutia extracts, received a significantly larger number of egg‐batches than control trees. In parallel, the volatiles emitted by all pine extracts tested where analysed by gas chromatography and mass spectrometry (GC‐MS), coupled to a time‐of‐flight (TOF) mass analyser after solid phase microextraction (SPME), resulting in the identification of 26 compounds. Comparative chromatograms showed qualitative differences among the pine species used, some compounds being present in one of them only. Findings demonstrate for the first time that: (i) T. pityocampa females discriminate among bouquets extracted from different host pine species and exhibit oviposition preferences; and (ii) olfactory cues play an important role in mediating the selection process.     

An allelopathic substance in red pine needles (Pinus densiflora)

Aqueous methanol extracts of red pine (Pinus densiflora) needles inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), ryegrass (Lolium multiflorum), timothy (Pheleum pratense), Digitaria sanguinalis and Echinochloa crus-galli. Increasing the extract concentration increased inhibition, suggesting that the pine needles may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of the pine needles was purified, and a main inhibitory substance was isolated and determined by spectral data as 9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli seedlings at concentrations greater than 0.1 mM. The endogenous concentration of the substance was 0.13 mmol/kg pine needle. These results suggest that 9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine.     

Connections between climatic variables and the growth and needle dynamics of Scots pine (Pinus sylvestris L.) in Estonia and Lapland

The relationships between climatic variables and Scots pine (Pinus sylvestris L.) growth and needle dynamics were studied in three stands in Estonia and in four stands located near the northern timberline in Lapland. The trees sampled in Estonia had low correlations with the analysed climatic variables (air temperature, precipitation and indices of atmospheric circulation). Moreover, the weak cross-correlation of the time-series of the Estonian sample trees indicated that Scots pine is affected mainly by local factors in that region. In Lapland, however, height increment and needle production correlated strongly among trees within a stand (mean r=0.45 and 0.46, respectively) and between stands (r=0.32 and 0.37). Radial increment also showed a high inter-correlation among the trees within a stand in Lapland (r=0.45). Both height increment and needle production were strongly influenced by the temperature regime of the previous summer in Lapland (mean r=0.64 and 0.64, respectively). Radial increment was correlated with the mean July temperature of the current year (mean r=0.29). The correlations between the indices of atmospheric circulation and tree attributes were weak, while the strongest correlation was between the Ponta Delgada NAO index (PD–NAO) and height increment and needle production in Lapland. Height increment, needle production and radial increment have increased since the 1990s in the trees growing in Lapland. This may indicate a positive effect of climate warming on tree growth in Lapland. In Estonia, where climatic conditions do not limit tree growth, the climate warming seems not to directly influence the growth and needle dynamics of Scots pine.     

Allelopathic potential of ground cover species onPinus resinosa seedlings

Summary Water extracts of foliage ofAster macrophyllus, Lonicera tatarica, Solanum dulcamara, Solidago gigantea, Prunus serotina, andRubus idaeus var.strigosus, collected from the ground vegetation of a red pine (Pinus resinosa Ait.) plantation, were tested for potential allelopathic effects on seed germination, radicle elongation, and growth of red pine seedlings. Seed germination was not significantly affected by extracts, but radicle elongation of newly germinated seeds was reduced by up to 48% compared to controls. All extracts variously inhibited height growth, formation of secondary needles, and dry weight increment of young red pine seedlings during a seven-week experiment. Total dry weight of seedlings treated with Lonicera extract was only 46% that of control plants. Phosphorus concentration in needles treated with Lonicera or Solidago extracts was significantly lower than that of controls. Growth inhibition was not mediated by pH effects or osmotic potentials of the extracts. The data suggest that allelopathic inhibition of red pine is possible, but additional field experiments are needed to evaluate the significance of allelopathy in red pine plantations.Research conducted by the College of Agricultural and Life Sciences. University of Wisconsin-Madison.     

High-performance liquid chromatography and micellar electrokinetic chromatography of taxol and related taxanes from bark and needle extracts of Taxus species

High-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC) were applied for the separation of taxol, cephalomannine, and baccatin III in crude extracts from the needle and bark of Taxus species. The chromatogram of the bark extract was cleaner than that of the needle allowing a more reliable detection of taxol and cephalomannine in the bark extract. However, HPLC quantitation of taxol in the needle extract would be difficult due to coeluting taxinines. Nevertheless, this was not a problem in the MEKC experiment. In comparison to HPLC, MEKC offered baseline resolution of taxol from taxinines in the needle extract, less solvent waste, a smaller sample requirement, and the simultaneous detection of taxol, cephalomannine and baccatin III in a relatively simpler electrophoretic run.     

Physiological investigations on the effect of olive and rosemary leaves extracts in male rats exposed to thioacetamide

Physiologically, it is known that thioacetamide (TAA) toxicity is generally associated with hepatic fibrosis induction, complicated metabolic disorders and health problems. The capability of extracts of olive and rosemary leaves to attenuate the severe physiological disturbances induced by thioacetamide (TAA) intoxication in male rats has been evaluated. Healthy male Wistar rats were used in the present study and were divided randomly into eight groups. Rats of the first group were served as normal control. Rats of the second group were administrated with TAA. Rats of the third, fourth and fifth groups were exposed to TAA plus olive leaves extract, TAA plus rosemary leaves extract and TAA plus olive and rosemary leaves extracts respectively. The sixth, seventh and eighth groups were supplemented with olive leaves extract, rosemary leaves extract, and olive and rosemary leaves extracts respectively. After 12 weeks of experimental treatments, the levels of serum glucose, total protein, albumin and high density lipoprotein cholesterol were significantly decreased, while the levels of triglycerides, cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, creatine kinase and lactate dehydrogenase were statistically increased in rats exposed to TAA. Administration of the studied extracts inhibited the hematobiochemical parameters and improved the physiological disturbances induced by TAA intoxication. Additionally, most improvements were noted in rats administrated with rosemary leaves extract followed by olive and rosemary leaves extracts and olive leaves extract. These results suggested that the effect of these extracts might be due to their antioxidant activities against TAA toxicity.     

Properties of Phenylalanyl-tRNA Synthetase and Phenylalanine Ammonia-lyase of Pine Suspension Cultures

Phenylalanyl-tRNA synthetase and phenylalanine ammonia-lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl-tRNA synthetase reaction was 7.5 and the optimum ATP and Mg²⁺ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia-lyase reaction was 9.0. The K_m for phenylalanine was approximately 6.6 × 10^?5M. The activity of both enzymes in the partially purified extracts was unstable on storage.